1. Field of the Invention
The present invention relates to an active biological molecule of the peptide type which when it is linked covalently to certain transporter peptides and/or certain adjuvants is capable of inducing in vivo a potentiating effect on the biological activity of growth hormone GH.
2. Description of the Related Art
The potentiation of the biological activity, more particularly of the somatogenic activity, of GH by antibodies was first observed by Holder et al. (1980), who based their studies on the measurement of incorporation of radioactive sulfates into costal cartilage as well as on the weight gain induced by GH in Snell type dwarf mice.
Three studies based on the same parameters performed by Holder et al. (1985) and Aston et al. (1986 and 1987) showed that the majority of the antibodies studied had a potentiating effect on the biological action of GH. The work of Wallis et al. (1987) confirmed the phenomenon of enhancement of the somatogenic activity of bovine growth hormone by anti-GH antibodies. These latter based their studies on the measurement of the IGF-1 (insulin growth factor) plasma level and the weight gain of hypophysectomized rats of the Wistar strain.
In particular, the European patent application EP 284 406 (COOPERS ANIMAL HEALTH LTD.) described a peptide having a primary structure homology with a continuous sequence of amino acid residues of the growth hormone in the region extending from positions 35 to 53 or peptides with cross-reactivity, said peptide being usable in an antigenic composition to potentiate the effects of growth hormone in a vertebrate. The experiments described by Ashton et al. consist of an in vivo administration of antibodies, either in the form of sera when the latter are obtained from sheep, or in the form of monoclonal antibodies and this passive administration of antibodies directed against the peptide or directed against native natural growth hormone, complexed with said hormone, increases the biological activity of the latter. It has not been demonstrated that the peptide 35-53 of growth hormone could be used in the framework of active immunization.
Recent studies performed with potentiating antibodies would seem to indicate that if GH alone binds to hepatocytes, the GH-MAb complex or the complex formed between the hormone according to the invention and a monoclonal potentiating antibody would bind preferentially to the sinusoidal cells (Kupffer cells) (Tans et al. (1994)). Thus, it might consequently be assumed that the synthesis of IGF-1 and IGF BP3 in the hepatocytes (Massart et al. (1993)) might also be enhanced by the action of said complexes at these sinusoidal cells.
It is described in the patent application EP 137 234 (THE WELLCOME FOUNDATION LIMITED) that certain antibodies to growth hormone are capable of potentiating the activity of the latter whereas it is known that, usually, such antibodies have a tendency to antagonize its action, at least in vivo. Such antibodies may in addition be produced in situ by the xe2x80x9cvaccinationxe2x80x9d of the host animal with a specific fragment of growth hormone, such that a class of polyclonal antibodies of restricted specificity is created which might potentiate the activity of the endogenous hormone.
In the present invention it is shown that certain sequences of growth hormone linked to a carrier molecule and/or an adjuvant are capable of potentiating the activity of said hormone in a vertebrate. The discovery of the peptide fragment of the present invention is the result of research based initially on the selection of monoclonal antibodies (MAb) of the IgG class, whose reactivities towards growth hormone, in particular bovine growth hormone, appeared to be the highest. After purification of said MAb, four types were selected. The effect of these latter on the plasma IGF-1 response induced by a single injection of bGH into the immature hypophysectomized rat was assayed by Massart et al. (1993). Finally, it was possible to identify an epitope recognized by a potentiating antibody designated 2H4, this antibody being capable of specifically recognizing a peptide sequence situated in the xcex1-helix No. 3 of native growth hormone.
Every reference to the primary, secondary or tertiary structure of GH refers to that described in Scanes C.G. et al. (1995).
The objective of the present invention is to provide a means for potentiating growth hormone GH more effectively and more durably than those of the prior art. In particular, the present invention provides a peptide or hapten capable of inducing in vivo a potentiating effect of the biological activity of this hormone when it is covalently linked to a transporter peptide and/or an adjuvant.
Such an effect may be the result of active immunization of the vaccination type, in which the administration of the hapten induces, in particular, the production of specific antibodies in vivo.
The present invention thus relates to a peptide construction comprising all or part of the sequence extending from position 103 to position 114 of the growth hormone GH or of a homologous peptide of said sequence provided that it exhibits an immunological cross-reactivity with said sequence, this peptide fragment being linked covalently to a transporter peptide and/or an adjuvant and being capable of inducing in vivo a potentiating effect on the biological activity of said growth hormone.
The precise identification of this GH sequence or xe2x80x9cGH peptidexe2x80x9d resulted from the analysis of a certain number of anti-GH monoclonal antibodies, responsible for a potentiating effect on the administration of the antibody-GH complex. This peptide fragment, the peptide sequence of which derived from GH may also be considered as a hapten-type molecule, is recognized by the antibody 2H4. Moreover, among the antibodies having the highest potentiating effects it may be noted that the affinity of 2H4 for GH is low compared with those of other antibodies.
According to a preferred peptide construction of the invention, said sequence of growth hormone GH is comprised in the following sequence:
GTSDRVYEKL SEQ ID NO:1.
Even more preferably, said sequence of the GH is selected from the following sequences:
TSDRVYEKL SEQ ID NO:2;
GTSDRVYEK SEQ ID NO:3;
SDRVYEKL SEQ ID NO:4;
TSDRVYEK SEQ ID NO:5; or
GTSDRVYE SEQ ID NO:6.
These five peptides correspond to the regions 105-113, 10-112, 106-113, 105-112, 104-111, respectively, of the growth hormone GH as published in Scanes et al. (1995) and may be obtained by the standard procedures of peptide synthesis.
Since a hapten is, by definition, non-immunogenic, the peptide of the invention is coupled covalently to a transporter. As examples, such transporters may advantageously be peptide sequences derived from ovalbumin, KLH (keyhole limpet hemocyanin) or albumin and more particularly the following peptides:
323-339 of ovalbumin
378-398 or 379-398 or 378-397 or 378-396 or 378-395 of the peptide CS.T3 described by Sinigaglia et al.,
45-60 of protein 1A of the respiratory syncitial virus
120-140 of the protein enveloping the genomic RNA of the hepatitis B virus.
These sequences are coupled covalently to the carboxyl or amino terminal of the xe2x80x9cGH peptidexe2x80x9d.
The peptide fragment of the invention may be linked covalently to an adjuvant of the N-acetyl-muramyl (MAP) type, linked to a peptide like MDP or its derivatives.
According to a preferred embodiment of the peptide fragment according to the invention, the adjuvant is the muramyl-dipeptide (MDP) or one of its derivatives such as MDP-lysine, Lys (NH2)xe2x80x94Dxe2x80x94isoGluxe2x80x94Lxe2x80x94Alaxe2x80x94NMcxe2x80x94Nur.
The MDP derivative is then coupled covalently to the carboxyl end of the peptide consisting of the xe2x80x9cGH peptidexe2x80x9d and the transporter, approximately similar to that described in EP 89290.
The peptide fragment according to the invention may also be linked to a sequence known or presumed to be T-dependent with or without MDP or its derivatives or alternatively linked or incorporated into liposomes. It may also be administered in aqueous or oily media, by the oral route, in the form of subcutaneous, intramuscular or transmucosal injections, by xe2x80x9cpelletsxe2x80x9d (biodegradable polymers containing the product) and by implantation of systems of dispersal by means of micropumps. Other adjuvants such as PAO alone or combined with lecithin (EP 445710), Zn(OH)2 or HBW 538 (DRUGS.EXP.CLIN RES. 17 (9) 1991 445-450) combined with Al(OH)3 may be linked covalently or not with the transporter GH peptide.
Finally, of course, several peptide fragments according to the invention may be bound to the same transporter molecule without the potentiating effect being affected.
The xe2x80x9cGH peptidesxe2x80x9d or GH peptides coupled to transporter peptides may be synthesized chemically, by methods known to the specialist skilled in the art, as for example the Merrified procedure.
Such an approach which implies active immunization is a procedure possessing many advantages compared with the administration of antibodies. In fact, active immunization, like vaccination, makes possible a durable effect whereas the administration of antibodies has only a transient effect; in addition, the production of peptides is appreciably less expensive than that of antibodies or of native GH.
Also included in the framework of the present invention is a recombinant nucleic acid incorporating a nucleotide sequence coding for the xe2x80x9cGH peptidexe2x80x9d according to the invention, preferably coupled to the peptide transporter. Such a recombinant nucleic acid may be used:
either to transfect prokaryotic or eukaryotic cells and make them produce said peptides in vitro;
or as active ingredient of a medicine potentiating GH; the peptides are then produced in vivo by a mechanism similar to that described in WANG, B. et al. (1993).
The present invention also relates to an immunogenic composition containing a peptide fragment or a recombinant nucleic acid described above.
An adjuvant may be added to such a composition, whether coupled to the peptide as described above or not. A composition according to the invention may advantageously be used as feedstuffs additive to stimulate the growth and/or lactation of animals, in particular cattle, sheep, pigs and other vertebrates (fish, marsupials, humans . . . ). It may be intended for oral administration, for example in the form of feedstuffs additive, or for rectal, subcutaneous, intramuscular or transmucosal administration, or by xe2x80x9cpelletsxe2x80x9d and micropump systems; in particular liposomes may be used as a vector in a composition incorporating the GH peptide, coupled or not to a transporter and an adjuvant.
For an effect designed to promote growth or lactation in animals, the composition is formulated such that the peptide of the invention is administered at a concentration of 0.01 xcexcg to 10 xcexcg per kg.
The peptides and the compositions of the invention may also be used in the case of humans in the manufacture of a medicine against growth disorders, whether the latter result from a deficit of endogenous growth hormone or from a disturbance of the metabolism of this latter. The formulations of these compositions would also be such as those defined above. Without being limiting, the examples and figures presented hereafter show the particularly advantageous effect of the peptide constructions of the invention, and particularly in comparison with other growth promoters.
In a preliminary step, it was necessary to select from among the different monoclonal antibodies directed against bGH those which exhibit a certain affinity for the latter. These latter seemed in fact to be those which would be the most likely to interfere positively or negatively with the biological activity of the hormone.
In order to obtain these antibodies a rapid immunization procedure described by Holmdahl et al. (1985) and Mirza et al. (1987) enabling specific monoclonal antibodies to be obtained was used. The antigen used was bGH.
The antibodies obtained were characterized with respect to:
isotypy
reactivity in RIA with bGH labelled with iodine 125
reactivity in ELISA (additivity titration, capture)
reactivities in WESTERN-BLOT
The antibodies were then purified from ascites by the procedure described in Bruck et al. (1982), and once the purification was completed, the solutions of antibodies obtained were subjected to mini-electrophoresis on a denaturing gel with SDS (PHAST SYSTEM). The purpose of this operation is to check the isolation of the immunoglobulins.
The capacity of the purified MAbs to form high concentrations of immune complexes with bGH in solution was determined by a method described in particular by Massart. This reactivity is measured by the capacity of the antibody to form a complex with I125 labelled bGH under precipitation conditions in the presence of PEG (polyethylene glycol).
This has enabled an antibody, designated 2H4 and exhibiting the desired reactivity, to be selected and studied with respect to its affinity for bGH. The results of the physico-chemical characteristics of this antibody are presented in the thesis of S. Massart and in Table 1 below.
React. Titr.: Titration of the anti-bGH MAb of the ascites fluids (represents the O.D. value observed ranging from 0.200 (1) to 2,000 (10)).
ELISA capt.: capture of biotinylated bGH with the aid of antibodies adsorbed on plastic (represents the number of antibody dilutions showing the observed maximal O.D.)
b) Mapping of the Epitope Recognized by the Antibody 2H4:
The work described above thus enabled the epitope of the MAb 2H4 to be defined by epitopic mapping studies performed in particular according to Beattie and Holder (1994).
A battery of octapeptides was synthesized and allowed to react with the monoclonal antibodies. The peptides which bind to the antibodies were identified by spectrometry. The molecular weight of the antibody-peptide complexes is markedly higher than that of the peptides alone.
Consequently, it was demonstrated that the antibody binding site was the peptide 104-113 of the structure of GH.
This region 104-113 is located in the xcex1-helix No. 3 of GH, a region of GH very little studied owing to the fact that the peptides of this helix exhibit a low radioreceptor activity (Scanes et al., Ed. 1995). However, it is known that the peptides of this region exhibit significant growth promoting activity, the xcex1-helix No. 3 is in addition the region of the second binding site of the GH binding protein (GHBP). In fact, for the GH to bind to its hepatic receptor, it is necessary that it constitutes a dimer with two GHBP in order to induce the growth promoting effect according to the model of Fuh (1992) (Science, 256, 1677-1678). In the absence of this dimerization of the GH or GHBP no antagonistic action is produced and thus no growth promoting effect is obtained. It may then be supposed that the potentiating antibody facilitates or modifies this GHBP binding at the binding site, which might explain the advantageous effect of this xe2x80x9cGH peptidexe2x80x9d.